1. Field of the Invention
This invention relates generally to immunoassays (particularly solid-phase fluorescent immunoassays--SPFIAs), to devices for detecting analytes by immunoassay using capillary tubes, to an apparatus for use with such devices, and to manual, semi-automated, and automated, methods for such testing.
2. Background of the Invention
Many situations exist where qualitative, semi-quantitative, or quantitative detection of the presence of an analyte in a sample is desired. Situations where analyte detection is desirable arise in diverse industries, including: 1) the health care industry, e.g., in clinical and diagnostic medicine (e.g., in vitro analysis); 2) the food processing and chemical industries, e.g. in quality control for food production; and 3) the environmental control industry, e.g. monitoring for the presence of various pollutants in air, ground water, or soil.
Many assays using unique devices and protocols to detect the presence of analytes through chemical and physical means have been developed. Immunoassays make up one broad field of assays which find use in the detection of analytes. In immunoassays, the occurrence of binding events between specific binding pair members is used as an indication of the presence of analyte in the sample. Benefits of using immunoassays, as compared to non-immunoassays, in analyte detection include high sensitivity, high specificity, reliability, and relatively short assay times.
The binding events that are utilized in immunoassays often occur at the surface of a solid support with one binding member held at the surface of the solid support and the other binding member in the sample. The time required for a particular immunoassay to be completed will depend on the ability of the binding member in the sample to reach and bind to the member on the support surface. The ability of the binding member in the sample to bind with its pair on the support surface is dependent on many factors; such factors include the concentration of the binding member on the support surface, and the surface to volume ratio of the sample/support combination. One method to decrease the time required for an immunoassay is to increase the concentration of a binding member on a support surface. Another approach is to increase the ratio of the surface area of the support relative to the volume of the sample to be assayed.
Common immunoassays include radio immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), and membrane based assays, such as common home pregnancy tests. These immunoassays have several disadvantages. A significant disadvantage of RIA is the requirement for the use of hazardous radioactive isotopes. A disadvantage of ELISA is the numerous steps of sample addition, incubation, washing, addition of color reagent, addition of stop reagent, and reading required to perform the assay; such manipulations can be especially troublesome and a source of significant error in the field. Also, enzyme reactions tend to be temperature sensitive, which require temperature control. Unlike RIA, in an which the label is directly detected, in an ELISA the enzyme label is not directly detected. Instead, one must allow for a detectable product to be produced. Further, ELISA protocols may not be suited for all assays on all types of liquids, such as where the liquid comprising the analyte of interest contains contaminants which interfere with one or more individual steps in the assay, e.g. enzyme activity, detectability of enzyme product, and the like. Additionally, the safety concerns with RIA and the complexity of ELISA typically require that they be performed by relatively highly trained personnel and further require constant monitoring by or interaction with the trained operator. A disadvantage with membrane based assays is that they often provide poor quantitation and sensitivity. Many of these assays also require long incubation times, typically in the tens of minutes to hours, making analysis of multiple samples time consuming and expensive.
Nevertheless, ELISA is a commonly used format. In ELISA, binding events of interest are detected through the appearance of detectable product produced by an enzyme acting on a substrate. The formation of the detectable product can be amplified to the extent required by increasing the concentration of the substrate and/or increasing the reaction time. On this basis, there is an opportunity to greatly increase the signal when only a few enzymes becoming bound.
Conventionally, ELISA has been conducted in microtiter plates consisting of wells. In an effort to improve performance, ELISA has been demonstrated in capillary tubes. With ELISA immunoassays conducted in capillary tubes, rapid quantitative results are reported. At least one reported ELISA is described as sensitive and able to detect small amounts of analyte. See e.g. Chandler et. al., "A new enzyme immunoassay system suitable for field use and its application in a snake venom detection kit" Clinica Chimica Acta, 121:225-230 (1982). However, the aforementioned disadvantages inherent in ELISA still exist.
Additionally, while ELISA assays have been performed in capillary tubes in the laboratory, no successful products have been developed. A primary reason for this is the difficulty of bringing several solutions into and out of the capillary tubes and the ability to effectively read the result.
Therefore, there is a significant need for a fast, reliable, accurate immunoassay that requires minimal interaction with the operator. There is also a need for an immunoassay that can screen several similar or different samples sequentially or simultaneously for the same analyte or which can screen for different analytes in the same sample or in a plurality of aliquots of the same sample.